Glycosyltransferase-catalyzed reactions have gained increasing attention and application for the synthesis of complex carbohydrates and glycoconjugates. Sialyltransferases, in particular, are the key enzymes that catalyze the transfer of a sialic acid residue from cytidine 5′-monophosphate-sialic acid (CMP-sialic acid) to an acceptor. Resulting sialic acid-containing products have been implicated in various biological and pathological processes, including cell-cell recognition, cell growth and differentiation, cancer metastasis, immunological regulation, as well as bacterial and viral infection. Besides being prevalent in mammals, sialyltransferases have been found in some pathogenic bacteria. They are mainly involved in the formation of sialic acid-containing capsular polysaccharides (CPS) and lipooligo(poly)saccharides (LOS/LPS), serving as virulence factors, preventing recognition by host's immune system, and modulating interactions with the environment. Sialyltransferases have been used for the synthesis of sialic acid-containing molecules with or without CMP-sialic acid biosynthetic enzymes (Li, Y. and Chen, X. 2012).
Cloning of sialyltransferases from various sources, including mammalian organs, bacteria, and viruses has been reported. Bacterial sialyltransferases have been cloned from several gram-negative bacteria belonging to Escherichia, Campylobacter, Neisseria, Photobacterium, Haemophilus, and Pasteurella genera. The genera Pasteurella and Haemophilus, both belong to the Haemophilus-Actinobacillus-Pasteurella (HAP) group, generally produced negatively charged outer cell surface and contain multiple genes encoding functional sialyltransferases. Two functional α2-3-sialyltransferases encoded by 1st and Hd0053 have been identified from Haemophilus ducreyi. Lic3A, SiaA, LsgB, and Lic3B are four sialyltransferases involved in the complex process of lipopolysaccharide sialylation in Haemophilus influenzae. Recently, a second sialyltransferase (PmST2) encoded by a Pm0508 gene homolog was characterized following the report on the first sialyltransferase from Pasteurella multocida encoded by a Pm0188 gene homolog (PmST1).
Most mammalian glycosyltransferases—including sialyltransferases—suffer from no or low expression in E. coli systems and more restricted substrate specificity. In comparison, bacterial glycosyltransferases are generally easier to access using E. coli expression systems and have more promiscuous substrate flexibility. Although certain wild-type bacterial glycosyltransferases with promiscuities for both donor and acceptor substrates have been discovered, readily obtainable enzymes with a wider substrate tolerance are needed to further the application of glycosyltransferases. The present invention meets this and other needs, providing surprisingly useful sialyltransferases for synthesis of glycoconjugates.